Selective cell therapy for the treatment of renal failure

ABSTRACT

Provided herein are isolated populations of kidney cells harvested from differentiated cells of the kidney, wherein the cells have been expanded in vitro. The kidney cells preferably produce erythropoietin (EPO). The kidney cells may also be selected based upon EPO production. Methods of producing an isolated population of EPO producing cells are also provided, and methods of treating a kidney disease resulting in decreased EPO production in a patient in need thereof are provided, including administering the population to the patient, whereby the cells express EPO in vivo in an oxygen tension-dependent manner.

RELATED APPLICATIONS

This application is a continuation-in-part and claims the benefit under 35 U.S.C. §120 of U.S. patent application Ser. No. 12/134,813, filed Jun. 6, 2008, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 60/942,716, filed Jun. 8, 2007, the disclosure of each of which is incorporated herein by reference in its entirety.

This application also claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 61/199,337, filed Nov. 14, 2008, the disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention is in the field of selective cell therapy for the restoration of organ function.

BACKGROUND OF THE INVENTION

Chronic renal failure is characterized by a gradual loss in kidney function, and may eventually progress to end stage renal failure, where the kidney no longer functions at a level to sustain the body. End stage renal failure is a devastating disease that involves multiple organs in affected individuals. The most common cause of end stage renal disease in the U.S. is diabetes.

One of the functions performed by the kidney is the production of erythropoietin (EPO). When the kidney is functioning properly, low tissue oxygenation in the renal interstitium stimulates the interstitial cells to produce EPO. The secreted EPO in turn stimulates red blood cell production in the bone marrow, which restores tissue oxygen tension to normal levels. Anemia caused by ineffective hematopoiesis is one of the inevitable outcomes of chronic renal failure due to the kidney's decreased ability to produce EPO. EPO has also been reported to protect against oxidative stress and apoptosis.

The kidney is the primary producer of EPO in the body and is therefore a primary target of treatment for renal failure induced anemia. Although dialysis can prolong survival for many patients with end stage renal disease, only renal transplantation can currently restore normal function. However, renal transplantation is severely limited by a critical donor shortage.

Treatments used to alleviate anemia associated with renal failure over the years include repeated transfusions of red blood cells and administration of testosterone and other anabolic steroids. However, none of these modalities has been entirely satisfactory. Patients receiving repeated transfusions are subject to iron overload, and may develop antibodies to major histocompatibility antigens. Testosterone has a minimal effect on erythropoeisis in the bone marrow, and it is associated with undesirable, virilizing side effects.

Previous efforts to mitigate anemia associated with renal failure have included the administration of purified recombinant EPO (See, e.g., U.S. Pat. Nos. 6,747,002 to Cheung et al., 6,784,154 to Westenfelder). However, the administration of recombinant EPO only elevates EPO levels in the blood temporarily, and may lead to iron deficiency. Gene therapy approaches have also been pursued, in which EPO is produced using transfected host cells (See, e.g., U.S. Pat. Nos. 5,994,127 to Selden et al., 5,952,226 to Aebischer et al., 6,777,205 to Carcagno et al.; Rinsch et al. (2002) Kidney International 62:1395-1401). However, these approaches involve the transfection of non-kidney cells, and require techniques such as cell encapsulation to prevent antigen recognition and immune rejection upon transplantation. Also, transfection with exogenous DNA may be unstable, and the cells may lose their ability to express EPO over time.

Renal cell-based approaches to the replacement of kidney tissue is limited by the need to identify and expand renal cells in sufficient quantities. In addition, the culturing of renal cells for the purpose of kidney tissue engineering is particularly difficult, owing to the kidney's unique structural and cellular heterogeneity. The kidney is a complex organ with multiple functions, including waste excretion, body homeostasis, electrolyte balance, solute transport, as well as hormone production.

There remains a great need for alternative treatment options to alleviate anemia caused by the failure of renal cells to produce sufficient amounts of erythropoietin.

SUMMARY OF THE INVENTION

Provided herein in embodiments of the present invention are isolated populations of kidney cells harvested from differentiated cells of the kidney that have been passaged and/or expanded in vitro. In some embodiments, the kidney cells include peritubular interstitial and/or endothelial cells of the kidney. In some embodiments, the kidney cells consist of or consist essentially of peritubular interstitial and/or endothelial cells of the kidney harvested from kidney tissue and passaged in vitro. In some embodiments, cells produce erythropoietin (EPO). In further embodiments, kidney cells are selected for EPO production. In some embodiments, the population has oxygen tension-dependent erythropoietin (EPO) expression.

Also provided are methods of producing an isolated population of EPO producing cells, including the steps of: 1) harvesting differentiated kidney cells; and 2) passaging the differentiated kidney cells, wherein the cells produce EPO after said passaging; thereby producing an isolated population of EPO producing cells. In some embodiments the methods further include the step of selecting the differentiated kidney cells for EPO production. In some embodiments, the passaging step includes growth of differentiated kidney cells in a medium comprising insulin transferrin selenium (ITS).

Methods of treating a kidney disease or other ailment, which disease or ailment results in decreased EPO production in a subject (e.g., a patient) in need thereof are also provided, including the steps of: 1) providing an isolated population of EPO producing cells; and 2) administering the population to the subject (e.g., in an amount effective to treat the kidney disease and/or the decreased EPO production), whereby the EPO producing cells produce EPO in vivo. In some embodiments, the providing step is performed by harvesting differentiated kidney cells of the kidney and passaging the cells in vitro. In some embodiments, the population of EPO producing cells includes, consists of or consists essentially of differentiated peritubular endothelial and/or interstitial cells harvested from differentiated cells of the kidney and passaged in vitro. In some embodiments, the population is provided in a suitable carrier (e.g., a collagen gel) for administration. In some embodiments, the administering step is carried out by implanting the population of cells into the kidney of the patient. In some embodiments, the administering step is carried out by subcutaneously injecting or implanting said composition. In some embodiments, the EPO producing cells are human. In some embodiments, the cells have oxygen tension-dependent EPO expression.

Further provided are isolated populations of cells including differentiated human kidney cells harvested from human kidney tissue and passaged in vitro. In some embodiments, the kidney cells consist of or consist essentially of peritubular interstitial and/or endothelial cells of the kidney harvested from kidney tissue and passaged in vitro. In some embodiments, the differentiated human kidney cells produce erythropoietin (EPO). In some embodiments, the human kidney cells have been passaged from 1-20 times. In some embodiments, the human kidney cells have been passaged at least 3 times. In some embodiments, the population has been selected for EPO production. Some embodiments are subject to the proviso that the cells are not transfected with an exogenous DNA encoding a polypeptide.

Compositions comprising the population of human kidney cells as described herein and a pharmaceutically acceptable carrier are also provided. In some embodiments, the carrier comprises collagen.

Also provided are methods of selecting cells for erythropoietin (EPO) production, including providing a composition comprising differentiated kidney cells, and selecting EPO producing cells from said composition, wherein said EPO producing cells have oxygen tension-dependent EPO expression. In some embodiments, the EPO producing cells have an increase in EPO expression as measured by an increase in EPO mRNA when under hypoxic conditions as compared to normoxic conditions, and have a decrease in EPO expression as measured by a decrease in EPO mRNA when cells return to normoxic conditions from hypoxic conditions.

Further provided are kits, including a media for growing differentiated kidney cells; an antibody or active fragment thereof which selectively binds EPO; and optionally, instructions for use.

Another aspect of the present invention is the use of the methods as described herein for the preparation of a composition or medicament for use in treatment or for carrying out a method of treatment as described herein (e.g., for treating a kidney disease or other ailment resulting in decreased EPO production), or for making an article of manufacture as described herein.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1. Mechanism of erythropoietin (EPO) production. Renal interstitial peritubular cells of the kidney detect low blood oxygen levels, and EPO is secreted into the blood. EPO stimulates the proliferation and differentiation of erythroid progenitors into reticulocytes, and prevents apoptosis, causing more reticulocytes to enter the circulating blood. The reticulocytes differentiate into erythrocytes, increasing the erythron size. Oxygen delivery to the tissues is thereby increased.

FIG. 2. Intracellular erythropoietin immunoreactivity was confirmed in the primary culture of renal cells at passage 1 (P1), passage 2 (P2) and passage 3 (P3), compared to the negative control (X400).

FIG. 3. Microscopy images of erythropoietin expressing cells in kidney tissue (left panel) and in cultured kidney cells (right panel).

FIG. 4. Quantification of erythropoietin (EPO) producing cells. The number of cells expressing EPO decreased with the subsequent passages (*p<0.05).

FIG. 5. Western blot analysis of detergent-solubilized cell extracts detected EPO protein (34 kDa) of early passage primary cultured renal cells (P0-P3).

FIG. 6. EPO expression analysis using FACS. Top Row: Mouse cells, passages 0-3. Bottom Row: Rat cells, passages 0-3.

FIG. 7A-7B. Mouse renal cell characterization. EPO expression is confirmed by immunofluorescence (FIG. 7A) (KNRK cells were used as positive control). GLEPP1 and Tamm Horsfall kidney markers were also detected (FIG. 7B).

FIG. 8. Rat renal cell characterization. Cultured rat kidney cells have various cell morphologies shown by phase contrast microscope (left panels), and express GLEPP1 and Tamm Horsfall kidney markers (right panels).

FIG. 9. EPO expression in HepG2 cells was shown by western blot and compared with EPO expression in kidney tissue.

FIG. 10. EPO protein expression of cultured cells under hypoxic conditions. Lewis rat kidney cells and HepG2 cells were cultured under normal and hypoxic conditions, and EPO production was assessed by western blot of cells. 34 kDa=EPO; 43 kDa=β-Actin.

FIG. 11. EPO protein expression in the culture medium under hypoxic conditions. EPO in the culture medium of Lewis rat kidney cells and HepG2 cells was assessed by western blot. 34 kDa=EPO; 43 kDa=β-Actin.

FIG. 12. Total protein lysates were prepared from rat renal primary cells at passages 1 and 2. Plates from normoxic samples (NC), samples in 3% O2 and 7% O2 were processed and run on 10% SDS-PAGE. KNRK cell line was used as positive control.

FIG. 13. Measuring EPO in media concentrates by western blot. Primary cultured cells from Lewis rats were raised close to confluency at each passage on 10 cm plates. The cells were starved with KSFM for 24 hrs and then placed in a hypoxic chamber (1% O2) for 24, 48 or 72 hrs. Following hypoxia incubation, the media was collected and concentrated with a 10K mwco amicon ultra centrifugal device (Millipore). 40 ug of total protein was then loaded on a 10% polyacrylamide gel. KNRK cells were used as positive control.

FIG. 14. Histological analysis of the retrieved implants showed that the kidney cells survived and formed tissue in vivo. Presence of EPO producing cells were confirmed immunohistochemically using EPO specific antibodies (×400). Left panel: Initial cell density of 1×10⁶ cells/injection. Right panel: Initial cell density of 1×10⁶ cells/injection. Top row of each panel: 2 weeks. Bottom row of each panel: 4 weeks.

FIG. 15. Effect of culture media and hypoxia on renal primary cells measured by real time PCR. Renal primary cells (p0) were grown to 80% confluency in 10 cm plates. Three plates of cells were grown with either serum free KSFM or DMEM and placed in a hypoxic chamber at 3% O2. After 24 hrs, samples were processed for total RNA and cDNA synthesis. Real time PCR was done in triplicate, and samples were quantified relative to normoxic sample.

FIG. 16. Effect of hypoxia on renal primary cells measured by real time PCR. Renal primary cells (passages 0 and 2) were grown to 80% confluency in 10 cm plates. Cells were then grown in serum free KSFM and placed in a hypoxic chamber at 1% O2. After 24, 48 or 72 hrs, samples were processed for total RNA and cDNA synthesis. Real time PCR was done in triplicate, and samples were quantified relative to normoxic sample.

FIG. 17. Effect of hypoxia on renal primary cells measured by real time PCR. Renal primary cells (passage 0) were grown to 80% confluency in 10 cm plates. Cells were then placed in a hypoxic chamber at 1% O2 for up to 24 hrs. Samples were then processed for total RNA and cDNA synthesis. Real time PCR was done in triplicate, and samples were quantified relative to normoxic sample

FIG. 18. Primary human kidney cells were expanded. Shown are cells of passages 2, 4, 7 and 9.

FIG. 19. Human primary renal cells were maintained through 20 doublings.

FIG. 20. Human kidney cell characterization. GLEPP1 and EPO positive cells are present in the population.

FIG. 21. Human kidney cell delivery in vivo with a 20 mg/ml collagen carrier. At retrieval, 3 weeks after injection, the injection volume had been maintained, and neovascularization was present.

FIG. 22. Injection of collagen with cultured human kidney cells resulted in EPO expressing tissue formation in vivo.

FIG. 23. Time course of EPO gene expression in rat primary renal cells under 1% O₂ continuous hypoxia. Cells were allowed to grow in culture to 70% confluency. After washing the cells and changing medium, the culture plates were placed in continuous hypoxia from 2 hrs up to 72 hrs. At end points, cells were processed for total RNA followed by relative EPO gene quantification using real-time per. Results are expressed relative to the gene expression amount of normoxia samples. Results given as mean+/−SEM (n=5).

FIG. 24. Effect of hypoxia and media change on EPO expression. Rat primary renal cells were placed in a 1% O₂ hypoxic chamber over 72 hrs. For some plates, media was changed once after 24 hrs or twice after 24 hrs and 48 hrs. All plates were processed for qPCR analysis of total RNA for EPO expression. Results given as mean+/−SEM (n=3).

FIG. 25. Effect of hypoxia and normal conditions on EPO expression. Rat primary renal cells were either placed in a 1% O₂ hypoxic chamber for up to 24 hrs or placed in a hypoxic chamber for 10 hrs followed by incubation in normoxia for 2, 4, and 8 hrs. All samples were processed for qPCR analysis of total RNA for EPO expression. Results given as mean+/−SEM (n=5).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Cell based therapy for renal failure can be approached in two directions: total and selective. Described herein is the selective cell therapy approach for achieving restoration of specific functional organ components.

The disclosures of all United States patent references cited herein are hereby incorporated by reference to the extent they are consistent with the disclosure set forth herein. As used herein in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the terms “about” and “approximately” as used herein when referring to a measurable value such as an amount of a compound, dose, time, temperature, and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount. Also, as used herein, “and/or” or “/” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

“Kidney tissue” is tissue isolated or harvested from the kidney, which tissue contains kidney cells. In some embodiments, kidney cells are positive for one or more known kidney markers, e.g., GLEPP1, Tamm Horsfall, etc. “Cell” or “cells” may be of any suitable species, and in some embodiments are of the same species as the subject into which tissues produced by the processes herein are implanted. Mammalian cells (including mouse, rat, dog, cat, monkey and human cells) are in some embodiments particularly preferred. “Isolated” as used herein signifies that the cells are placed into conditions other than their natural environment. Tissue or cells are “harvested” when initially isolated from a subject, e.g., a primary explant. Harvesting of kidney tissue may be performed in accordance with methods known in the art. See also U.S. Patent Application Publication No. 2004/0167634 (Atala et al.), which is incorporated by reference herein.

“Subjects” are generally mammalian, including human, subjects and include, but are not limited to, “patients.” The subjects may be male or female and human subjects may be of any race or ethnicity, including, but not limited to, Caucasian, African-American, African, Asian, Hispanic, Indian, etc. The subjects may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric.

Subjects and patients may also include animal subjects, particularly mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, non-human primates, etc., for, e.g., veterinary medicine and/or pharmaceutical drug development purposes.

Cells may be syngeneic (i.e., genetically identical or closely related, so as to minimize tissue transplant rejection), allogeneic (i.e., from a non-genetically identical member of the same species) or xenogeneic (i.e., from a member of a different species). Syngeneic cells include those that are autogeneic (i.e., from the patient to be treated) and isogeneic (i.e., a genetically identical but different subject, e.g., from an identical twin). Cells may be obtained from, e.g., a donor (either living or cadaveric) or derived from an established cell strain or cell line. Cells may be harvested from a donor, e.g., using standard biopsy techniques known in the art.

The “primary culture” is the first culture to become established after seeding disaggregated cells or primary explants into a culture vessel. “Expanding” as used herein refers to an increase in number of viable cells. Expanding may be accomplished by, e.g., “growing” the cells through one or more cell cycles, wherein at least a portion of the cells divide to produce additional cells.

“Passaged in vitro” or “passaged” refers to the transfer or subculture of a cell culture to a second culture vessel, usually implying mechanical or enzymatic disaggregation, reseeding, and often division into two or more daughter cultures, depending upon the rate of proliferation. If the population is selected for a particular genotype or phenotype, the culture becomes a “cell strain” upon subculture, i.e., the culture is homogeneous and possesses desirable characteristics (e.g., the ability to express EPO).

“Express” or “expression” of EPO means that a gene encoding EPO is transcribed, and preferably, translated. Typically, according to the present invention, expression of an EPO coding region will result in production of the encoded polypeptide, such that the cell is an “EPO producing cell.” In some embodiments, cells produce EPO without further manipulation such as the introduction of an exogenous gene. In some embodiments, the invention is subject to the proviso that the EPO producing cells are not manipulated by the introduction of an exogenous gene and/or by an exogenous chemical that stimulates the production of EPO.

In some embodiments, EPO expression is “oxygen-dependent” in that the level of EPO expression changes in an oxygen concentration-dependent or oxygen tension-dependent manner (the oxygen concentration or oxygen tension being that of the surrounding media/carrier/tissues/bodily fluids which are in fluid contact with the cells). Kidney cells isolated, cultured and/or administered according to some embodiments which have oxygen-dependent EPO expression show an upregulation (increase) of EPO expression when under hypoxic conditions (e.g., 1% 09) as compared to normoxic conditions as found in vivo and/or under cell culture conditions commonly represented in the art in vitro by incubation in 5% CO₂ and 95% atmospheric air. EPO expression is also downregulated (decreased) when cells return to normoxic conditions.

For example, in some embodiments when the kidney cells are at 70% confluency, they may have an oxygen-dependent increase in EPO expression as measured by an increase in EPO mRNA expression (e.g., by 2, 4, 8, 10 or 20-fold) after incubation for 24 hours under hypoxic conditions of: a humidified incubator at 37° C., at atmospheric pressure, and with 1% O₂, 5% CO₂ and 94% N₂; as compared to normoxic conditions of: a humidified incubator at 37° C., at atmospheric pressure, and with 5% CO₂ and 95% atmospheric air. The incubation media may be a media described herein, e.g., a 1:1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco's Modified Eagle's Medium (DMEM) based media, with the DMEM based media being ¾ DMEM and ¼ HAM's F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100×, 1 ml of 0.4 m/ml hydrocortisone, 0.5 ml of a 10⁻¹⁰ M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μg/ml epidermal growth factor (EGF) solution. Preferably, the cells have been washed with said incubation media and fresh incubation media applied (e.g., within one hour prior to said incubation for 24 hours) in order to prevent possible negative feedback due to the presence of excreted EPO.

In some embodiments, harvested cells are not passaged. In other embodiments, cells are passaged once, twice, or three times. In still other embodiments, cells are passaged more than 3 times. In some embodiments, cells are passaged 0-1, 0-2 or 0-3 times. In some embodiments, cells are passaged 1-2, 1-3, or 1-4 or more times. In some embodiments, cells are passaged 2-3 or 2-4 or more times. In further embodiments, cells are passaged 5, 8, 10, 12 or 15 or more times. In some embodiments, cells are passaged 0, 1, 2, 3 or 4 to 8, 10, 15 or 20 or more times. The number of passages used may be selected by, e.g., the relative EPO production measured in the cell population after each passage.

Growing and expansion of kidney cells is particularly challenging because these cells are prone to the cessation of growth and early differentiation. This challenge is overcome in some embodiments of the present invention by using kidney cell specific media that contains additives that promote their growth. Accordingly, in some embodiments kidney cells are grown in media that includes additives such as growth factors and other supplements that promote their growth. Further, in some embodiments, EPO producing cells are grown in co-culture with other renal cell types.

In some embodiments, kidney cells are grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) or fetal calf serum (FCS) and, optionally, penicillin-streptomycin (P/S). In other embodiments, kidney cells are grown in keratinocyte serum-free medium (KSFM). In further embodiments, kidney cells are grown in KSFM with one or more of the following additives: bovine pituitary extract (BPE) (e.g., 50 g/mL), epidermal growth factor (EGF) (e.g., 5 ng/mL), antibiotic-antimycotic solution (GIBCO) (e.g., 5 mL), fetal bovine serum (FBS) (Gemini Bio-Product) (e.g., 12.5 mL of 2.5%), and insulin transferrin selenium (ITS) (Roche) (e.g., 50 mg for 5 L medium). As understood by those of skill in the art, in some embodiments of the above media, penicillin-streptomycin (P/S) and antibiotic-antimycotic solution are interchangeable.

In some embodiments, kidney cell are grown in media that is a 1:1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco's Modified Eagle's Medium (DMEM) based media. The premixed DMEM based media according to some embodiments is ¾ DMEM and ¼ HAM's F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100×, 1 ml of 0.4 μg/ml hydrocortisone, 0.5 ml of a 10⁻¹⁰ M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μg/ml epidermal growth factor (EGF) solution.

Passaging of kidney cells according to some embodiments may be accomplished using standard procedures known in the art. For example, the cells may be detached using trypsin/EDTA and transferred to other plates. This is a standard procedure for many cell types. Briefly, in some embodiments this may be accomplished with the following steps: 1) Remove medium. 2) Add 10 ml PBS/EDTA (0.5 M) for 4 minutes. Confirm the separation of cell junctions under a phase contrast microscope. 3) Remove PBS/EDTA and add 7 ml Trypsin/EDTA. 4) Add 5 ml medium when 80-90% of the cells lift under microscope. 5) Aspirate the cell suspension into a 15 ml test tube. 6) Centrifuge the cells at 1000 rpm for 4 minutes. 7) Remove the supernatant. 8) Resuspend cells in 5 ml of medium. 9) Pipet out 100 μl of the cell suspension and perform trypan blue stain for viability assay. 10) Count the number of cells on hemocytometer. 11) Aliquot the desired number of cells on the plate and make the volume of medium to a total of 10 ml. 12) Place the cells in the incubator.

“Selection” can be based upon any unique properties that distinguish one cell type from another, e.g., density, size, unique markers, unique metabolic pathways, nutritional requirements, protein expression, protein excretion, etc. For example, cells may be selected based on density and size with the use of centrifugal gradients. Unique markers may be selected with fluorescent activated cell sorting (FASC), immunomagnetic bead sorting, magnetic activated cell sorting (MASC), panning, etc. Unique metabolic pathways and nutritional requirements may be exploited by varying the makeup and/or quantity of nutritional ingredients of the medium on which cells are grown, particularly in a serum-free environment. Protein expression and/or excretion may be detected with various assays, e.g., ELISA.

“EPO producing cell” refers to differentiated cells, of which at least a portion produce EPO (e.g., at least 20, 30, 40, or 50% or more, or more preferably 60, 70, 80, or 90% or more of the cells produce EPO). In some embodiments, cells produce EPO without further manipulation such as the introduction of an exogenous gene. In some embodiments, the invention is subject to the proviso that the EPO producing cells are not manipulated by the introduction of an exogenous gene and/or by an exogenous chemical that stimulates the production of EPO. The cells may be harvested from, e.g., the peritubular interstitial cells of the kidney. In some embodiments, the cells are selected for their ability to produce EPO. In other embodiments, the cells are expanded in number by cell culture techniques, e.g., passaging. Cells with the specific function of EPO production can be used from the kidney and from other sources. For example, EPO is also normally produced in the liver.

In the kidney, EPO is generally known to be produced by the interstitial peritubular cells (FIG. 1). In some embodiments, an isolated population of differentiated kidney cells comprises, consists of or consists essentially of interstitial peritubular cells of the kidney, consisting of or consisting essentially of 80, 90, 95, or 99 percent or more, or not more than 20, 10, 5 or 1 percent or less, by number of other cell types. In other embodiments, the isolated population of differentiated kidney cells includes other cell types, e.g., endothelial peritubular cells.

In some embodiments, the isolated population of differentiated kidney cells comprises, consists of or consists essentially of kidney cells that are selected for EPO production, consisting of or consisting essentially of 80, 90, 95, or 99 percent or more, or not more than 20, 10, 5 or 1 percent or less, by number of cells not expressing EPO. Selection may be accomplished by selecting the cells that express EPO using specific markers. In some embodiments, cells may include various types of kidney cells, so long as the cells express EPO. In further embodiments, the entire renal cell colony may be used for expansion and treatment.

In some embodiments, the isolated population of differentiated kidney cells have a “longevity” such that they are capable of growing through at least 5, 10, 15, 20, 25 or 30 or more population doublings when grown in vitro. In some embodiments, the cells are capable of proliferating through 40, 50 or 60 population doublings or more when grown in vitro.

“Differentiated” refers to cells or a population containing cells that have specialized functions, e.g., EPO production and/or expression of known markers of differentiated cells (e.g., GLEPP1 and/or Tamm Horsfall kidney cell markers). In this sense they are not progenitor or stem cells. Some embodiments of the present invention are subject to the proviso that harvested differentiated cells are not passaged under conditions to create a population of less specialized cells.

Alternatively, in other embodiments, cells are cultured to produce cell lines, which may later be differentiated to produce more specialized cells. The establishment of “cell lines,” as opposed to cell strains, are by and large undifferentiated, though they may be committed to a particular lineage. Propagation naturally favors the proliferative phenotype, and in some embodiments cells may require a reinduction of differentiation by, e.g., alteration of the culture conditions. There are a number of differentiation factors known in the art that may induce differentiation in cell lines (e.g., cytokines such as epimorphin and HGF, vitamins, etc.).

Methods of Treatment.

In some embodiments, EPO producing cells are administered to a subject in need thereof (e.g., by injection) to the kidney (e.g., into the cortex and/or medulla). In other embodiments, EPO producing cells are administered to other areas of the body, e.g., the liver, peritoneum, etc. In some embodiments, the EPO producing cells are administered subcutaneously, subcapsular, etc. In further embodiments, EPO producing cells are administered by implantation of a substrate (e.g., a collagen gel scaffold) containing said EPO producing cells described herein. In still other embodiments, EPO producing cells are administered through vascular access (e.g., systemically or locally).

Diseases that may be treated with the methods disclosed herein include, but are not limited to, anemias. Anemias include, but are not limited to, those associated with renal failure or end-stage renal disease, anemias caused by chemotherapies or radiation, anemias of chronic disorders, e.g., chronic infections, autoimmune diseases, rheumatoid arthritis, AIDS, malignancies, anemia of prematurity, anemia of hypothyroidism, anemia of malnutrition (e.g., iron deficiency), and anemias associated with blood disorders.

“Treat” refers to any type of treatment that imparts a benefit to a patient, e.g., a patient afflicted with or at risk for developing a disease (e.g., kidney disease, anemia, etc.). Treating includes actions taken and actions refrained from being taken for the purpose of improving the condition of the patient (e.g., the relief of one or more symptoms), delay in the onset or progression of the disease, etc.

Other endocrine systems may benefit from the therapies disclosed herein, for example, vitamin D producing cell therapy or the angiotensin system. See, e.g., U.S. Patent Application Publication No. 2005/0002915 to Atala et al., which is incorporated herein by reference. Cells with a specific function can be used from the kidney and other sources, i.e., cells that would produce target functions. For example, EPO is also normally produced in the liver.

Preferably the cells are mixed with or seeded onto a pharmaceutically acceptable carrier prior to administration. “Pharmaceutically acceptable” means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment. Such formulations can be prepared using techniques well known in the art. See, e.g., U.S. Patent Application 2003/0180289; Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, Pa., 2000. The carrier may be a solid or a liquid, or both (e.g., hydrogels), and can be formulated with the cells as a unit-dose formulation. In some embodiments the cells are provided as a suspension in the carrier to reduce clumping of the cells. In other embodiments cells are seeded onto a biodegradable scaffold or matrix.

In some embodiments, cells are mixed with a suitable gel for administration. Suitable gels that may be used in the present invention include, but are not limited to, agars, collagen, fibrin, hydrogels, etc. Besides gels, other support compounds may also be utilized in the present invention. Extracellular matrix analogs, for example, may be combined with support gels to optimize or functionalize the gel. One or more growth factors may also be introduced into the cell suspensions. See also U.S. Patent Application Publication No. 2007/0116679 (Atala), which is incorporated by reference herein.

Formulations of the invention include those for parenteral administration (e.g., subcutaneous, intramuscular, intradermal, intravenous, intraarterial, intraperitoneal injection) by injection or implantation. In one embodiment, administration is carried out intravascularly, either by simple injection, or by injection through a catheter positioned in a suitable blood vessel, such as a renal artery. In some embodiments, administration is carried out by “infusion,” whereby compositions are introduced into the body through a vein (e.g., the portal vein). In another embodiment, administration is carried out as a graft to an organ or tissue to be augmented as discussed above, e.g., kidney and/or liver.

A “biodegradable scaffold or matrix” is any substance not having toxic or injurious effects on biological function and is capable of being broken down into is elemental components by a host. Preferably, the scaffold or matrix is porous to allow for cell deposition both on and in the pores of the matrix. Such formulations can be prepared by supplying at least one cell population to a biodegradable scaffold to seed the cell population on and/or into the scaffold. The seeded scaffold may then implanted in the body of a recipient subject.

In some embodiments, cells are administered by injection of the cells (e.g., in a suitable carrier) directly into the tissue of a subject. For example, cells may be injected into the kidney (e.g., the subcapsular space of the kidney). Because the functional effects of EPO production will be systemic, cells may also be administered by injection into other tissues (e.g., the liver, subcutaneously, etc.).

Cells may also be delivered systemically. In further embodiments, cells are delivered to tissue outside of the kidney (e.g., the liver), as the outcome of the functional effects of EPO production will be systemic. See, e.g., the “Edmonton protocol,” an established delivery method, where cells are infused into a patient's portal vein (Shapiro et al. (2000) N Engl J Med 343:230-238).

According to some embodiments, the cells administered to the subject may be syngeneic (i.e., genetically identical or closely related, so as to minimize tissue transplant rejection), allogeneic (i.e., from a non-genetically identical member of the same species) or xenogeneic (i.e., from a member of a different species), as above, with respect to the subject being treated, depending upon other steps such as the presence or absence of encapsulation or the administration of immune suppression therapy of the cells. Syngeneic cells include those that are autogeneic (i.e., from the subject to be treated) and isogeneic (i.e., a genetically identical but different subject, e.g., from an identical twin). Cells may be obtained from, e.g., a donor (either living or cadaveric) or derived from an established cell strain or cell line. As an example of a method that can be used to obtain cells from a donor (e.g., a potential recipient of a bioscaffold graft), standard biopsy techniques known in the art may be employed. Alternatively, cells may be harvested from the subject, expanded/selected in vitro, and reintroduced into the same subject (i.e., autogeneic).

In some embodiments, cells are administered in a therapeutically effective amount. The therapeutically effective dosage of cells will vary somewhat from subject to subject, and will depend upon factors such as the age, weight, and condition of the subject and the route of delivery. Such dosages can be determined in accordance with procedures known to those skilled in the art. In general, in some embodiments, a dosage of 1×10⁵, 1×10⁶ or 5×10⁶ up to 1×10⁷, 1×10⁸ or 1×10⁹ cells or more per subject may be given, administered together at a single time or given as several subdivided administrations. In other embodiments, a dosage of between 1-100×10⁸ cells per kilogram subject body weight can be given, administered together at a single time or given as several subdivided administration. Of course, follow-up administrations may be given if necessary.

Cells may be administered according to some embodiments to achieve a target hematocrit range. The ideal or target hematocrit range may vary from subject to subject, depending upon, e.g., specific comorbidities. In some embodiments the target hematocrit is from 30-40%, in some embodiments the target hematocrit is from 33-38%, and in some embodiments the target hematocrit is from 33-36%. Upon administration of cells according to the present invention, hematocrit may be measured and, if desired or necessary, corrected by, e.g., further implantation of cells and/or other methods known in the art (e.g., supplementing with recombinant EPO). Other methods of treatment for anemia and/or renal disease may be used in conjunction with the methods of treatment provided herein, for example, an adapted protein-caloric intake diet.

In further embodiments, if desired or necessary, the subject may be administered an agent for inhibiting transplant rejection of the administered cells, such as rapamycin, azathioprine, corticosteroids, cyclosporin and/or FK506, in accordance with known techniques. See, e.g., R. Calne, U.S. Pat. Nos. 5,461,058, 5,403,833 and 5,100,899; see also U.S. Pat. Nos. 6,455,518, 6,346,243 and 5,321,043. Some embodiments use a combination of implantation and immunosuppression, which minimizes graft rejection. The implantation may be repeated as needed to create an adequate mass of transplanted tissue.

The present invention is explained in greater detail in the following non-limiting Examples.

EXAMPLES

Anemia is an inevitable outcome of chronic renal failure due to the kidney's decreased ability to produce erythropoietin (EPO) by peritubular interstitial cells. We investigated whether supplementation of erythropoietin producing cells would be a possible treatment option for renal failure-induced anemia by examining the feasibility of selecting and expanding erythropoietin producing cells for cell-based therapy.

The following examples demonstrate that EPO producing cells are present in renal cells harvested from mouse and rat kidneys. In addition, cells isolated and expanded using the methods described below include cells expressing EPO at every culture stage examined. Further, the actual percentage of cells expressing the EPO marker in culture was consistent with the cell population present in normal kidney tissues (see Yamaguchi-Yamada et al., J Vet Med Sci, 67: 891, 2005; Sasaki et al., Biosci Biotechnol Biochem, 64: 1775, 2000; Krantz, Blood, 77: 419, 1991).

Example 1 Expansion of Renal Cell Primary Cultures

Renal cells from 7-10 day old mice C57BL/6 were culture expanded. Minced kidney (1 kidney of mouse) was placed into a 50 cc tube with 15 ml of collagenase/dispase (0.2 mg/ml). The kidney tissue fragments were incubated in a 37° C. shaker for 30 min with collagenase/dispase mix (0.2 mg/ml; 15 ml). Sterile PBS with Gelatin (20 ml), was added (with Gelatin (DIFCO) 2 mg/ml) to the digestion solution. The mixture was filtered thorough a 70 micron filter to remove undigested tissue fragments. The collected solution was mixed well (being careful not to make air bubbles), and divided into two 50 cc tubes. The tubes were centrifuged at 1000(−1500) RPM for 5 min. The supernatant was discarded and the pellet of each tube was resuspended in 3 ml of KSFM medium. DMEM medium (10% FBS, 5 ml P/S) is used for stromal cells, and KSFM with BPE, EGF, 5 ml antibiotic-antimycotic, 12.5 ml FBS (Gemini Bio-Product, 2.5%), Insulin Transferrin Selenium (Roche) (50 mg for 5 L medium) with BPE and EGF for epithelial components. P/S or antibiotic-antimycotic (GIBCO) may also be added. Each tissue was seeded on to a 25 mm plate and medium was added (total 3 ml).

Cells were maintained by changing the medium the next day, and then every 2 days depending on the cell density. Cells were passaged when they were 80-90% confluent by detachment using trypsin/EDTA and transferred to other plates with the following steps: 1) Remove medium. 2) Add 10 ml PBS/EDTA (0.5 M) for 4 minutes. Confirm the separation of cell junctions under a phase contrast microscope. 3) Remove PBS/EDTA and add 7 ml Trypsin/EDTA. 4) Add 5 ml medium when 80-90% of the cells lift under microscope. 5) Aspirate the cell suspension into a 15 ml test tube. 6) Centrifuge the cells at 1000 rpm for 4 minutes. 7) Remove the supernatant. 8) Resuspend cells in 5 ml of medium. 9) Pipet out 100 μl of the cell suspension and perform trypan blue stain for viability assay. 10) Count the number of cells on hemocytometer. 11) Aliquot the desired number of cells on the plate and make the volume of medium to a total of 10 ml. 12) Place the cells in the incubator.

Alternatively, the following protocol was used. Kidneys from 10 day old male C57BL/6 mice were collected in Krebs buffer solution (Sigma Aldrich, St. Louis, Mo. USA) containing 10% antibiotic/antimycotic (Gibco Invitrogen, Carlsbad, Calif. USA) to avoid risk of contamination. The kidneys were immediately transported to a culture hood where the capsule was removed. The medullary region of the kidney was removed, and only the cortical tissue was used to isolate cells that had been previously identified as EPO producing cells (Maxwell et al., Kidney International, 44: 1149, 1993). The kidney tissue was minced and enzymatically digested using Liberase Blendzyme (Roche, Mannheim, Germany) for 25 minutes at 37 degrees Celsius in a shaking water bath. The supernatant was removed and the cell pellet was passed through a 100 μm cell strainer to obtain a single cell suspension for culture.

Subsequently, the cells were plated at a density of 5×10⁵ cells/ml in 10 cm tissue culture treated plates filled with culture media. The culture media consisted of a mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco's Modified Eagle's Medium (DMEM) at a ratio of 1:1. The premixed DMEM media contained ¾ DMEM and ¼ HAM's F12 nutrient mixture supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100× (Gibco), 1 ml of 0.4 μg/ml hydrocortisone, 0.5 ml of a 10⁻¹⁰ M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μg/ml epidermal growth factor (EGF) solution. All tissue culture reagents were purchased from Sigma-Aldrich (St. Louis, Mo. USA) unless otherwise stated. The cells were incubated at 37° C. under 5% CO₂ with medium change every 3 days, and the cells were subcultured for expansion at a ratio of 1:3 when confluent.

Example 2 Characterization for EPO Production

The cells from early passages (1, 2 and 3) were characterized for EPO expression using immunocytochemistry and western blot analysis with specific antibodies (rabbit polyclonal anti-EPO antibodies, sc-7956, Santa Cruz Technologies, Santa Cruz, Calif.).

Renal cells were plated in 8-well chamber slides at a density of 3000 cells per well. The cells were incubated at 37° C. under 5% CO₂ for 24 h to allow attachment. This was followed by fixation with 4% paraformaldehyde for 10 minutes at room temperature. Permeabilization of cell membranes was performed by adding 0.1% Triton-X 100 in PBS for 3 minutes at room temperature. Cells were then incubated in goat serum for 30 minutes at room temperature. After washing, cells were incubated with the primary antibodies for 1 h (1:50) at room temperature. Cells were washed a second time and biotinylated goat polyclonal anti-rabbit antibodies (polyclonal anti rabbit IgG, Vector Laboratories, Inc., Burlingame, Calif.) (1:200) were added, followed by incubation at room temperature for 45 minutes. Chromogenic detection of EPO followed a final washing step and was performed using the Vector ABC kit according to the manufacturer's instructions (Vector Laboratories, Inc., Burlingame, Calif.). Slides without the primary antibodies served as internal negative controls, and normal mouse renal tissue served as the positive control.

Renal cells in culture showed multiple phenotypes under the microscope. The cells reached confluency within 7 to 10 days of plating. Many of the cells observed in the first 3 passages after isolation from the kidney stained positively for EPO, as compared to the negative controls, which showed no background or nonspecific staining (FIG. 2), which indicated that the observed staining was likely due to the presence of EPO in the cultures. The number of cells that stained positively for EPO remained constant throughout the 3 passages studied, even when phenotypic changes were observed in the culture during the same time period. Immunohistochemical staining of kidney tissue indicated a similar amount of EPO expression as that found in cultured cells (FIG. 3).

The number of cells expressing EPO decreased slightly with subsequent passages (FIG. 4). This is most likely due to the increased number of passages and loss of cells/function over time and manipulation. However, the relative percentage appears to remain stable after the first passage.

EPO expression was also confirmed by western blot, shown in FIG. 5.

Example 3 Mouse and Rat Renal Cell Characterization

FACS analysis was used to quantify the number of EPO-producing cells in the established renal cell cultures at each passage (1-3 passages). The cells were collected by trypsinization and centrifugation, resuspended in media, and passed through a 70 μm cell strainer to ensure a single cell suspension. After counting the cells, they were spun down and resuspended in PBS at 5-7.5×10⁵ cells/tube to remove FBS from the cells. The cells were fixed with 2% formaldehyde for 10 minutes at 4° C. and permeabilized using 100% methanol for 10 minutes at room temperature. Subsequently, the cells were resuspended in 3% goat serum in PBS followed by incubation with the rabbit anti-EPO primary antibody sc-7956 (Santa Cruz Biotechnology, Santa Cruz, Calif.) for 45 minutes on ice. Cells were washed twice with 3% goat serum in PBS prior to incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibodies for 1 hour. The cells were then washed thoroughly with 3% serum in PBS and transferred to the FACS machine (FACS Calibur E6204, Becton-Dickinson, Franklin Lakes, N.J.).

Fluorescent activated cell sorting experiments demonstrated that 44% of passage 1 (P1) cells were EPO positive. This percentage increased to 82% at passage 2 (P2), and then dropped back to 42% at passage 3 (P3). This may indicate that, during the first few days of culture, proliferation of EPO-producing cells and/or upregulation of EPO gene expression occurs in response to the lower oxygen concentration in the media compared to normal living tissue. These responses could then normalize over the next few days, resulting in numbers of EPO-producing cells that are close to those found in renal tissue (FIG. 6, top row).

The FACS data demonstrate the maintenance of EPO expression over several passages. It should be noted that there was a surge in the number of cells expressing EPO (82%) in the passage 2 culture, which was confirmed by several repeat experiments. Though not wishing to be bound to any particular theory, one possible explanation for this phenomenon could be that EPO expression is an inherent trait of all renal cells that can be turned on and off as needed. In this case, following the abrupt change in survival conditions between the body and the culture plate, the cells may have been driven to express EPO momentarily until stabilization of the culture occurred. Consistent with this, the EPO surge was quickly reversed and passage 3 analyses showed a lower percentage of EPO producing cells (42%).

Mouse cell characterization by immunofluorescence confirmed EPO expression (FIG. 7A). The population of cells was positive for the kidney cell markers GLEPP1 and Tamm Horsfall (FIG. 7B).

Rat cell passages 0, 1 and 2 were also analyzed for EPO production using fluorescence activated cell sorting (FACS) (FIG. 6, bottom row). Cultured rat cells had various cell morphologies and were positive for GLEPP1 and Tamm Horsfall kidney cell markers (FIG. 8).

Example 4 Exposure of EPO Producing Cultures to Hypoxic Conditions

While maintenance of phenotypic characteristics is essential during cell expansion stages, a critical component that ensures the success of cell therapy is the ability of EPO producing cells to regulate and maintain normal EPO levels. EPO belongs to the hematopoietic cytokine family, and it controls erythropoiesis in bone marrow, and regulates the proliferation, differentiation and survival of erythroid progenitor cells through EPO receptor (EPOR)-mediated signal transduction. EPO is largely produced in the kidney, and when this organ fails, EPO production falls, leading to anemia. EPO expression in the body depends largely on the oxygen tension in the environment surrounding the cells capable of producing EPO. Factors influencing oxygen levels include lack of oxygen in the ambient air and decreased renal blood flow.

To determine whether the EPO expressing cells in culture could respond to changing oxygen levels, an experiment was performed in which the cells were serum-starved for 24 hours followed by exposing them to various levels of oxygen in vitro. Lewis rat kidney cells and HepG2 (human hepatocellular liver carcinoma cell line) cells were cultured under normal and hypoxic conditions, and EPO production was assessed and confirmed by western blot of cells. EPO presence in the culture medium was also measured and confirmed by analyzing the supernatants from cultured renal cells under normoxic and hypoxic conditions with the double antibody sandwich enzyme-linked immunosorberbent assay using a Quantikine® IVD Erythropoietin ELISA kit (R&D Systems®, Minneapolis, Minn.).

The cells were placed in serum free media for 24 hours prior to the experiment. The plates were then transferred to a hypoxic chamber and exposed to different hypoxic conditions (1%, 3%, 5%, and 7% oxygen). HepG2 cells were used as positive controls, as they have been previously reported to produce high levels of EPO in culture (Horiguchi et al., Blood, 96: 3743). EPO expression by HepG2 was confirmed by western blot (FIG. 9). All cells were harvested in lysis buffer containing NP-40. Protein concentration in each sample was measured using a Bio-Rad protein assay. 40 ng total protein was run out on a 10% acrylamide gel using SDS-PAGE. Proteins were then transferred onto a PVDF membrane (Millipore Corp.). Detection of β-actin expression in the lysates was used as the loading control. EPO antibody (rabbit polyclonal sc-7956, Santa Cruz Biotechnology) was used at 1:200 and the secondary antibody (goat anti-rabbit 7074, Cell Signaling Technology, Beverly, Mass.) was used at 1:2000. To measure the amount of EPO secreted into the media by the primary renal cultures, the media was collected and concentrated down to 500 ul using an Amicon Ultra centrifugal filter device (Millipore Corporation, Billerica, Mass.). Samples of this media were run on a 10% polyacrylamide gel. EPO antibody (rabbit polyclonal sc-7956, Santa Cruz Biotechnology) was used at 1:100 and the secondary antibody (goat anti-rabbit 7074, Cell Signaling Technology, Beverly, Mass., USA) was used at 1:2000.

Western blotting showed a slight increase in the EPO expression in the cell lysate after hypoxia (FIG. 10). These results, however, were not seen when media concentrates were used to measure EPO (FIG. 11). The media testing indicated that all media concentrates (hypoxic and normoxic conditions) contained the same low amount of EPO.

Alternatively, total protein lysates were prepared from rat renal primary cells at passages 1 and 2. Plates from normoxic samples (NC), samples in 3% O2 and 7% O2 were processed and Run on 10% SDS-PAGE. The KNRK cell line was used as positive control. Results are shown in FIG. 12.

Without wishing to be bound by any particular theory, this may indicate that 24 hours might not be enough time for secreted EPO levels to rise to a level that is detectable by western blot. It is likely that a longer exposure time would be required for the cells to begin to secrete EPO, as de novo protein production may take several hours to become apparent. Therefore the following experiment was performed, in which cells were placed in hypoxic conditions for 24, 48 and 72 hours.

Primary cultured cells from Lewis rats were raised close to confluency at each passage on 10 cm plates. The cells were placed in a hypoxic chamber (1% O₂) for 24, 48 or 72 hrs. Following hypoxia incubation, the media was collected and concentrated with a 10K molecular weight cutoff Amicon Ultra centrifugal device (Millipore). 40 μg of total protein was then loaded on a 10% Polyacrylamide gel. KNRK cells were used as a positive control. Results are shown in FIG. 13.

In summary, all experiments indicated that the EPO levels in primary culture cells were greater than or equal to those measured in the HepG2 positive controls, and the EPO producing cells are able to respond to changing environment.

Example 5 Administration of EPO Producing Cells in vivo

To determine whether EPO producing cells survive and form the tissues in vivo, renal cells mixed in collagen gel were implanted subcutaneously in athymic mice at concentrations of 1×10⁶ and 5×10⁶ followed by retrieval at 14 and 28 days after implantation for analysis. Cells at different passages from 1-5 were used. The cells were suspended in a collagen gel for easy injection (concentration: 0.1 mg/ml).

Histologically, the retrieved implants showed that surviving renal cells continue expressing EPO proteins, confirmed immunohistochemically using EPO specific antibodies (FIG. 14).

These results demonstrate that EPO producing renal cells grown and expanded in culture stably expressed EPO in vivo. Thus, EPO producing cells may be used as a treatment option for anemia caused by chronic renal failure.

Example 6 Analysis of EPO Expression with Real Time PCR

Real time PCR was performed to assess rat cell expression of EPO in response to hypoxic conditions.

To test the effect of culture media, cells grown in either KSFM and DMEM were exposed to hypoxic conditions (3% O₂). Renal primary cells (passage 0) were grown to 80% confluency in 10 cm plates. Three plates of cells were grown with either serum free KSFM or DMEM and placed in a hypoxic chamber at 3% O₂. After 24 hrs, samples were processed for total RNA and cDNA synthesis. Real time PCR was done in triplicate, and samples were quantified relative to normoxic sample. Results are shown in FIG. 15.

Rat kidney culture EPO expression was compared with real time PCR across 24, 48 and 72 hours. Renal primary cells (passages 0 and 2) were grown to 80% confluency in 10 cm plates. Cells were then grown in serum free KSFM and placed in a hypoxic chamber at 1% O2. After 24, 48 or 72 hours, samples were processed for total RNA and cDNA synthesis. Real time PCR was done in triplicate, and samples were quantified relative to normoxic sample. Results are shown in FIG. 16.

Testing timepoints for up to 24 hours, renal primary cells (passage 0) were grown to 80% confluency in 10 cm plates. Cells were then placed in a hypoxic chamber at 1% O2 for up to 24 hours. Samples were then processed for total RNA and cDNA synthesis. Real time PCR was run in triplicate, and samples were quantified relative to normoxic sample. Results are shown in FIG. 17.

Example 7 Expansion of Human Kidney Cells

The growth and expandability of primary human kidney cells were also demonstrated using the media and conditions described above. Cultures from passages 2, 4, 7 and 9 are shown in FIG. 18. It was demonstrated that human primary renal cells can be maintained through twenty doublings (FIG. 19). Human kidney cell cultures were characterized for EPO and GLEPP1 expression (FIG. 20).

Example 8 Human Kidney Cell Delivery Via Collagen Injection

Human renal cells mixed in collagen gel were implanted subcutaneously in athymic mice as described above in Example 5. Collagen concentrations of 1 mg/ml, 2 mg/ml and 20 mg/ml were compared. At 1 and 2 mg/ml, the in vivo volume disappeared after administration. At 20 mg/mL, the in vivo injection volume was maintained, and neo-vascularization was seen FIG. 21. Histology confirmed that EPO expressing tissue was formed in vivo (FIG. 22).

Example 9 EPO Producing Cell Selection with Magnetic Cell Sorting

Cells are selected for EPO production using magnetic cell sorting. A single-cell suspension is isolated using a standard preparation method. After preparation of single-cell suspension, count the total number of the cells and centrifuge cell samples to obtain a pellet. Block the cells with 10% of goat serum (of animal where the secondary antibody is made) for 10 minutes. Add 1 or 2 mL of the blocking solution. After 10 minutes of centrifugation, resuspend the cells in the primary antibody for EPO (use 1 μg of the primary antibody/million of cells). Typically, label for 15 minutes at 4-8° C. is sufficient. Wash the cells twice to remove any unbound primary antibody with 1-2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 minutes. After two successive washes, the pellet is resuspended in 80 μL of PBS (0.5% of BSA and 2 mM of EDTA, pH 7.2) per 10⁷ cells. Add 20 μL of Goat Anti-Rabbit MicroBeads per 10⁷ cells. Mix well and incubate for 15 minutes at 4-8° C. Wash the cells twice by adding 1-2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 minutes. Pipette off supernatant completely. Resuspend up to 10⁸ cells in 500 μl, of buffer (Note: For higher cell numbers, scale up buffer volume accordingly; for depletion with LD Columns, resuspend cell pellet in 500 μL of buffer for up to 1.25×10⁸ cells).

Proceed to Magnetic Cell Separation

Note: Work fast, keep cells cold, and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and non-specific cell labeling. Volumes for magnetic labeling given below are for up to 10⁷ total cells. When working with fewer than 10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g. for 2×10⁷ total cells, use twice the volume of all indicated reagent volumes and total volumes). Working on ice may require increased incubation times. Higher temperatures and/or longer incubation times lead to non-specific cell labeling.

Example 10 Controlled Regulation of Erythropoietin Producing Cells for Renal Failure Induced Anemia

In this study we investigated whether production of EPO could be controlled by exposing EPO producing cells to different environmental conditions.

Methods: Renal cells from 2-3 week old rats were isolated, expanded and characterized for EPO expression using immunocytochemistry, FACS, and Western Blot analyses. To assess the levels of EPO expression, cells were incubated under normoxic and hypoxic (1%) conditions for varying time periods up to 24 hours. Another group of cells was subjected to alternating cycles of hypoxia and normoxia. To determine whether the presence of EPO in culture media influence EPO production, renal cells were grown in conditions either with or without media changes. All cells were collected at the end of each experiment for assessment using Western blot analysis and RT-PCR.

Results: Immunocytochemical analysis of the cultured renal cells showed the expression of EPO at each subculture stage (P1-P3). Western Blot analysis of the detergent-solubilized cell extracts detected EPO (34 kDa) protein in the kidney cells of all passages tested. RT-PCR analysis of renal cells exposed to hypoxia showed up-regulation of EPO gene expression over a period of 24 hours followed by a gradual decrease in the next 48 hours. However, this decrease was reversed completely when culture media was changed every 24 hours, which indicates their ability to regulate EPO production through a negative feedback mechanism. Decrease in EPO expression was also observed when cells under hypoxia were transferred to normoxic conditions. When these cells were placed back to hypoxic environment, the levels of EPO expression began to increase.

Conclusions: These results demonstrate that EPO producing renal cells possess the ability to regulate EPO expression in response to varying levels of oxygen. The cells readily respond to the amounts of surrounding EPO by either inhibiting or promoting EPO production. These findings indicate that EPO producing renal cells may be used as a treatment option for renal failure induced anemia.

Example 11 Hypoxia Induced Regulation of Erythropoietin Expression and Secretion in Primary Renal Cells

The examples provided above describe the identification, expansion and characterization of EPO-producing cells derived from kidney tissue. In this study, we show that these cells are able to regulate EPO expression and production when exposed to different oxygen concentrations. These results confirm that the cell-based therapy utilizing isolated EPO producing cells as disclosed herein may be used to correct anemia and are able to self-regulate EPO expression.

Erythropoietin (EPO) is the primary regulator of red blood cell production. EPO production is induced when the amount of oxygen to the tissues is reduced, and dysfunction in this feedback system can cause anemia. For patients with severe chronic kidney disease (CKD), anemia occurs when the EPO-producing cells in the damaged kidneys become non-functional.

Erythropoietin gene expression is primarily triggered by reduction in oxygen tension in renal tissues. Many circumstances can produce a reduction in oxygen tension, including anemia, high altitudes, and persistent blood loss. The method by which renal cells react to decreased oxygen tension and subsequently regulate the EPO gene expression is highly complex. The process involves a large number of regulatory molecules such as the hypoxia inducing factor (HIF1α) and other intracellular pathways (Eschbach JW (1989) Kidney Int 35, 134-148). The activation of these processes leads to the increased translation of the EPO gene, followed by release of the hormone into the bloodstream. The hormone binds to its receptors on erythroid precursors in the bone marrow to increase the production of new red blood cells (RBC). The increased RBCs circulating in the blood can then carry more oxygen to tissues, leading to another cascade of events that culminates in the down-regulation of the EPO gene.

In end stage renal disease (ESRD), the kidney becomes unable to produce enough EPO, and this leads to anemia. Currently, ESRD-related anemia is mainly treated with recombinant human EPO (rhuEPO) injections several times a week. However, the need for repeated injections is inconvenient for patients. In addition, accurate delivery of the proper rhuEPO dose is required, since high concentrations of this hormone could lead to polycythemia and the unwanted complications associated with it.

Our previous results showed a significant increase in EPO gene expression in cells exposed to hypoxia relative to those in normoxic conditions. However, the elevated levels of EPO expression decreased after 24 hrs. If cells were returned to normoxic conditions during the first 24 hours, a sharp decline in EPO expression occurred. In contrast, EPO excretion into the media in hypoxic and normoxic samples was unchanged during the initial 24 hrs, but was slightly elevated over 72 hours in samples under hypoxia.

As described below, we have found that refreshing the media of hypoxic samples every 24 hrs elicited up-regulation of EPO expression, indicating either sensitivity to feedback inhibition or apoptosis inhibition.

Rat primary renal cells were isolated using enzymatic digestion as described above. Briefly, kidneys of 2-week-old pre-weaning female Lewis rats were harvested and placed in a solution of PBS containing 1% antimycotic/antibiotic solution (Hyclone). The renal capsule was removed, and the kidney tissue was minced. Tissue pieces were placed in a Krebs buffer solution containing Liberase Blendzyme (Roche, Mannheim, Germany). Tissue was enzymatically digested for 1 hour in a 37° C. shaking water bath. The solution was then filtered through a 100 micron cell strainer (Becton Dickson, Franklin Lakes, N.J., USA) and centrifuged. Cells were suspended in culture media consisting of a mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco's Modified Eagle's Medium (DMEM) based media at a ratio of 1:1. The premixed DMEM-based media contained ¾ DMEM and ¼ HAM's F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100× (Gibco), 1 ml of 0.4 μg/ml hydrocortisone, 0.5 ml of a 10⁻¹⁰ M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μg/ml epidermal growth factor (EGF) solution. All tissue culture reagents were purchased from Sigma-Aldrich unless otherwise stated.

Cells from two kidneys were seeded in 10 cm tissue culture treated plates and cultured in normoxic conditions in a humidified incubator (37° C., 5% CO₂ and 95% atmospheric air). The cells attached to the cell culture plate after 2-3 days, and the cultures appeared to contain heterogenous renal cell types. The cells were cultured until they reached 70% confluency before transported into hypoxic conditions for the subsequent experiments. During serial passaging, the morphology of the rat cells became more similar to cells from a fibroblastic lineage by the third passage.

To determine the percentage of cells in the cultures that expressed EPO protein, we performed flow cytometry. In the initial culture (P0), in which cells were more heterogeneous, 56% of cells stained positive for EPO compared to 87% and 95% at passages 1 and 2, in which cells became progressively more homogeneous.

EPO expression in continuous hypoxia. Cells were subjected to hypoxia (1% O₂) over 72 hours. EPO mRNA transcript levels were analyzed at 2, 4, 6, 8, 12, 24, 48, and 72 h by real time PCR and compared to cells cultured in normoxic conditions. EPO gene expression increased significantly (8-fold) in cells under hypoxia for 24 hrs, then fell sharply after 24 hours, but did not reach the low levels of EPO expression observed in normoxic cells (FIG. 23).

EPO expression in continuous hypoxia with media change. We compared cells exposed to continuous hypoxia for up to 72 hours without medium replacement with cells that received fresh medium either once (at 24 hrs) or twice (at 24 hrs and 48 hrs) during the 72 hour culture period. After 48 hours, EPO mRNA expression was elevated significantly in cultures that received fresh medium. We observed a similar trend after 72 hours (FIG. 24). Changing the medium twice induced up-regulation of EPO mRNA significantly when compared to cells that did not receive fresh medium during the same period.

Effect of normoxia after exposure to hypoxic environment. To determine the effect of oxygen level normalization on EPO gene expression in hypoxic cells, we placed multiple plates in the hypoxic chamber for up to 24 hours. Plates were kept under hypoxic conditions for 24 hours, and then moved back to normal conditions for 2, 6, or 24 hours. This normoxic period was followed by another 2 or 6 hours in the hypoxic chamber. Other plates were moved back to normal conditions after 10 hours in hypoxia and RNA was isolated at 2, 4, and 8 hours in normoxia. Samples that were moved between hypoxic and normoxic conditions showed an EPO mRNA expression corresponding to changes in their environment, decreasing expression when moved to the normoxic environment and increasing expression when moved back to hypoxic conditions. When cells were moved back to normoxic conditions after 10 hrs of hypoxia, EPO mRNA expression fell relative to cells in continuous hypoxia (FIG. 25).

Effect of hypoxia on EPO secretion. In vivo, sensing of hypoxia in the tissues initiates a cascade of events that includes the up-regulation of EPO mRNA expression in the kidney and the eventual secretion of the EPO hormone into the bloodstream (3). We tested this phenomenon in vitro by culturing primary renal cells with conditioned medium without serum and placing them in a hypoxic chamber for 72 hrs. We then concentrated the spent media and performed an ELISA assay to measure EPO protein secreted into this media. Severe hypoxia (1% 09) did not significantly influence EPO secretion in the initial 24 hr period, although we have shown that EPO mRNA levels are increased during this time.

In this study, we demonstrated that primary renal culture cells regulate EPO expression according to the surrounding environment. We isolated, cultured and expanded rat primary renal cells to examine the regulation of EPO as a consequence of fluctuations in oxygen tension. As a proof of concept, we only used primary renal cells at passage ‘0’ to ensure observation of the most robust response.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. 

1. An isolated population of cells comprising differentiated kidney cells passaged in vitro, wherein said population has oxygen tension-dependent erythropoietin (EPO) expression.
 2. The population of cells according to claim 1, wherein said cells have an increase in EPO expression as measured by an increase in EPO mRNA when under hypoxic conditions as compared to normoxic conditions, and have a decrease in EPO expression as measured by a decrease in EPO mRNA expression when cells return to normoxic conditions from hypoxic conditions.
 3. The population of cells according to claim 1, wherein said cells have an increase in EPO expression as measured by a 2- to 20-fold increase in EPO mRNA after incubation of said cells at 70% confluencey for 24 hours: under hypoxic conditions of: a humidified incubator at 37° C., at atmospheric pressure, and with 1% O₂, 5% CO₂ and 94% N₂, as compared to incubation under normoxic conditions of: a humidified incubator at 37° C., at atmospheric pressure, and with 5% CO₂ and 95% atmospheric air, wherein said incubation media is a 1:1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbeeco's Modified Eagle's Medium (DMEM) based media, wherein said DMEM based media is ¾ DMEM and ¼ HAM's F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100×, 1 ml of 0.4 μg/ml hydrocortisone, 0.5 ml of a 10⁻¹⁰ M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μg/ml epidermal growth factor (EGF) solution, and wherein said cells have been washed with said incubation media and fresh incubation media applied within one hour prior to said incubation for 24 hours.
 4. The population of cells according to claim 1, wherein said cells have a decrease in EPO expression as measured by a 2- to 20-fold decrease in EPO mRNA after incubation of said cells at 70% confluency for 20 hours: under hypoxic conditions for 10 hours, said hypoxic conditions being: a humidified incubator at 37° C., at atmospheric pressure, and with 1% O₂, 5% CO₂ and 94% N₂, followed by incubation for 10 hours under normoxic conditions of: a humidified incubator at 37° C., at atmospheric pressure, and with 5% CO₂ and 95% atmospheric air; as compared to incubation for 20 hours under said hypoxic conditions given above, wherein said incubation media is a 1:1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco's Modified Eagle's Medium (DMEM) based media, wherein said DMEM based media is ¾ DMEM and ¼ HAM's F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100×, 1 ml of 0.4 μg/ml hydrocortisone, 0.5 ml of a 10⁻¹⁰ M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μg/ml epidermal growth factor (EGF) solution, and wherein said cells have been washed with said incubation media and fresh incubation media applied within one hour prior to said incubation for 20 hours.
 5. The population of cells according to claim 1, wherein said population has been passaged from 1 to 20 times.
 6. The population of cells according to claim 1, wherein said population has been passaged from 1 to 10 times.
 7. The population of cells according to claim 1, wherein said population has been passaged from 1 to 5 times.
 8. The population of cells according to claim 1, wherein said population has been selected for EPO production.
 9. The population of cells according to claim 1, subject to the proviso that said cells are not transfected with an exogenous DNA encoding a polypeptide.
 10. A composition comprising the population of cells according to claim 1 and a pharmaceutically acceptable carrier.
 11. A method of treating a kidney disease resulting in decreased erythropoietin (EPO) production in a patient in need thereof, said method comprising: providing a composition comprising differentiated kidney cells and a pharmaceutically acceptable carrier; and administering said composition to said patient, wherein said cells have oxygen tension-dependent EPO expression.
 12. The method of claim 11, wherein said pharmaceutically acceptable carrier comprises a collagen gel.
 13. The method of claim 11, wherein said cells have been passaged from 1 to 20 times prior to said administering.
 14. The method of claim 11, wherein said cells have been passaged from 1 to 10 times prior to said administering.
 15. The method of claim 11, wherein said cells have been passaged from 1 to 5 times prior to said administering.
 16. The method of claim 11, wherein said cells have been selected for EPO production prior to said administering.
 17. The method of claim 11, subject to the proviso that said cells are not transfected with an exogenous DNA encoding a polypeptide.
 18. The method of claim 11, wherein said administering is carried but by injecting said composition into the kidney or liver of said patient.
 19. The method of claim 11, wherein said administering step is carried out by injecting or infusing said composition intravascularly.
 20. The method of claim 11, where said administering step is carried out by infusing said composition into a portal vein of said patient.
 21. The method of claim 11, wherein said pharmaceutically acceptable carrier comprises a biodegradable scaffold administering step is carried out by implanting said composition into the kidney of said patient.
 22. The method of claim 11, wherein said kidney disease is an anemia selected from the group consisting of: an anemia of renal failure, an anemia of end-stage renal disease, an anemia of a chemotherapy, an anemia of a radiation therapy, an anemia of chronic infection, an anemia of an autoimmune disease, an anemia of rheumatoid arthritis, an anemia of AIDS, an anemia of a malignancy, an anemia of prematurity, an anemia of hypothyroidism, an anemia of malnutrition, and an anemia of a blood disorder.
 23. The method of claim 11, wherein said EPO producing cells are mammalian.
 24. The method of claim 11, wherein said EPO producing cells are human.
 25. A method of treating anemia in a patient in need thereof, said method comprising: providing a composition comprising the population of cells according to claim 10; and administering said composition to said patient in an amount effective to treat said anemia.
 26. The method of claim 25, wherein said administering step is carried out by injecting or implanting said composition into said patient.
 27. The method of claim 25, wherein said anemia is selected from the group consisting of: an anemia of renal failure, an anemia of end-stage renal disease, an anemia of a chemotherapy, an anemia of a radiation therapy, an anemia of chronic infection, an anemia of an autoimmune disease, an anemia of rheumatoid arthritis, an anemia of AIDS, an anemia of a malignancy, an anemia of prematurity, an anemia of hypothyroidism, an anemia of malnutrition, and an anemia of a blood disorder.
 28. A method of selecting cells for erythropoietin (EPO) production comprising: providing a composition comprising differentiated kidney cells; and selecting EPO producing cells from said composition, wherein said EPO producing cells have oxygen tension-dependent EPO expression.
 29. The method of claim 28, wherein said EPO producing cells have an increase in EPO expression as measured by an increase in EPO mRNA when under hypoxic conditions as compared to normoxic conditions, and have a decrease in EPO expression as measured by a decrease in EPO mRNA when cells return to normoxic conditions from hypoxic conditions.
 30. The method of claim 28, wherein said EPO producing cells have an increase in EPO expression as measured by a 2- to 20-fold increase in EPO mRNA after incubation of said cells at 70% confluencey for 24 hours: under hypoxic conditions of: a humidified incubator at 37° C., at atmospheric pressure, and with 1% O₂, 5% CO₂ and 94% N₂, as compared to incubation under normoxic conditions of: a humidified incubator at 37° C., at atmospheric pressure, and with 5% CO₂ and 95% atmospheric air, wherein said incubation media is a 1:1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco's Modified Eagle's Medium (DMEM) based media, wherein said DMEM based media is ¾ DMEM and ¼ HAM's F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100×, 1 ml of 0.4 μg/ml hydrocortisone, 0.5 ml of a 10⁻¹⁰ M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μg/ml epidermal growth factor (EGF) solution, and wherein said cells have been washed with said incubation media and fresh incubation media applied within one hour prior to said incubation for 24 hours.
 31. The method of claim 28, wherein said EPO producing cells have a decrease in EPO expression as measured by a 2- to 20-fold decrease in EPO mRNA after incubation of said cells at 70% confluency for 20 hours: under hypoxic conditions for 10 hours, said hypoxic conditions being: a humidified incubator at 37° C., at atmospheric pressure, and with 1% O₂, 5% CO₂ and 94% N₂, followed by incubation for 10 hours under normoxic conditions of: a humidified incubator at 37° C., at atmospheric pressure, and with 5% CO₂ and 95% atmospheric air; as compared to incubation for 20 hours under said hypoxic conditions given above, wherein said incubation media is a 1:1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco's Modified Eagle's Medium (DMEM) based media, wherein said DMEM based media is ¾ DMEM and ¼ HAM's F12 nutrient mixture supplemented with 5% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 1% glutamine 100×, 1 ml of 0.4 μg/ml hydrocortisone, 0.5 ml of a 10⁻¹⁰ M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μg/ml epidermal growth factor (EGF) solution, and wherein said cells have been washed with said incubation media and fresh incubation media applied within one hour prior to said incubation for 20 hours.
 32. A kit comprising: a media for growing differentiated kidney cells; an antibody or active fragment thereof which selectively binds EPO; and optionally, instructions for use.
 33. The kit of claim 32, wherein said media comprises a 1:1 mixture of keratinocyte serum-free medium (KSFM) and premixed Dulbecco's Modified Eagle's Medium (DMEM) based media, wherein said DMEM based media is % DMEM and ¼ HAM's F12 nutrient mixture supplemented with 5% serum, 1% antibiotic, 1% glutamine 100×, 1 ml of 0.4 μg/ml hydrocortisone, 0.5 ml of a 10⁻¹⁰ M cholera toxin solution, 0.5 ml of a 5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adenine solution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyronine mixture, and 0.5 ml of a 10 μg/ml epidermal growth factor (EGF) solution. 